Dendritic cells are now understood to provide two essential functions in the immune response. In their immature state they capture antigens and can promote tolerance, while in their mature state they stimulate T cells to mount effective immune responses. Maturation is mediated by a variety of stimuli, primarily provided by the pro-inflammatory products produced by microbial pathogens. The current program project application will exploit the novel approaches developed by this research team during the last funding period to test the hypothesis that distinct DC subsets mediate tolerance through distinct mechanisms. These subsets, defined by the CDS, DEC205 and 33D1 surface markers, contribute to tolerance through their ability to lead to the deletion or anergy of autoreactive T cells (the intrinsic pathway) or through their ability to promote the differentiation and expansion of CD25+CD4+foxp3+ regulatory T cells. The collective goals of the program project will be the 1) Manipulation of DC subsets to promote differentiation and expansion of antigen specific CD25+CD4+foxp3+ Treg cells in vivo;2) Regulation FcR signaling on DC subsets as a means of controlling DC maturation and the generation of antigen-specific tolerance and 3) Identification of the role of the DEC205 and 33D1 DC subsets in antigen presentation and tolerance induction. These studies will be pursued by an experienced team of senior investigators with a long history of successful collaborations and productivity, using novel approaches to target DC subsets in vivo and manipulate their maturation. These in vivo approaches with model antigens will then be applied to a series of autoimmune models through the targeting of relevant autoantigens to distinct DC subsets in an effort to restore antigen specific tolerance. The program will be augmented by three core facilities for FACS analysis and sorting, protein expression and autoimmune animal models to promote the research activities of the group. The knowledge gained from this integrated, systematic approach to the function of DC subsets in vivo will provide the basis for the design of novel therapeutic approaches to the treatment of autoimmune disorders. PROJECT 1: Dendritic cells induce tolerance to pancreatic islets (Project Leader: Steinman, Ralph) DESCRIPTION (provided by applicant): To silence autoimmune disease, we seek a means to expand in mice CD25+ CD4+ foxp3+ regulatory T cells (Treg) that are specific for disease producing autoantigens. We will pursue three themes that have begun through synergisms with Drs. Nussenzweig and Ravetch in this program: 1) The efficiency of antigen presentation in vivo can be greatly increased by targeted delivery of antigen within monoclonal antibodies to uptake receptors on dendritic cell (DCs). 2) A pivotal feature to the outcome of antigen presentation is the state of differentiation or maturation of the DC;this can be enhanced or blocked through selective ligation of activating and inhibitory Fc?R respectively. 3) DCs are specialized antigen presenting cells for CD25+ foxp3+ Treg, being able to drive their expansion with maintenance of foxp3 expression and function (5) and to convert CD25- foxp3- T cells into CD25+ foxp3+ T reg (preliminary data). Therefore we will identify DC receptors, subsets and maturation states that are required to control the development and maintenance of antigen-specific T reg in the peripheral tissues of mice. Aim 1 will determine the DC requirements for the expansion of CD25+ CD4+ foxp3+ antigen-specific Treg in vivo, pursuing initial evidence that the 33D1 receptor on CDS- DCs is an effective pathway. Aim 2 will determine the DC requirements for the differentiation of CD25+ CD4+ foxp3+ antigen-specific Treg in vivo from CD25- CD4+ foxp3- progenitors, pursuing initial evidence that the DEC-205 receptor on CD8+ DCs is an effective pathway and that TGFp works in concert with antigen presenting DCs to generate typical antigen-specific foxp3+ T reg. Aim 3 will induce, expand and maintain antigen-specific Tregs from a naive polyclonal T cell repertoire, including capacity of DC-targeting antibodies to ligate activating and inhibitory Fey receptors. By harnessing the biology of DCs in vivo, particularly by targeting antigens within monoclonal antibodies to uptake receptors expressed on DCs, and by controlling the state of differentiation or maturation of DCs, we will be able to define the principles for generating large numbers of disease specific regulatory T cells in intact mice. This should in turn translate into new targeted therapies and products required to control autoimmunity in patients in a much more disease specific manner than has previously been feasible.